Epstein-Barr virus (EBV) replication is accomplished by an intricate cascade that integrates DNA replication with structural protein expression. It has long been known that some EBV genes (called late genes) are dependent on viral DNA replication for their transcription, but the molecular basis for this dependency was unknown. The Johannsen lab has recently shown that a virally encoded pre-initiation complex (vPIC) requires an origin of lytic DNA replication to mediate late gene expression. This suggests a model where vPIC mediates recruitment of RNA polymerase 2 to newly replicated DNA in order to transcribe late genes. We have demonstrated that late gene transcripts co-localize with the EBV DNA replication compartments that have been intensively characterized by the Sugden lab. Remarkably, the Sugden group has shown that these compartments lack histone proteins, suggesting that vPIC mediates transcription of late genes from a non-chromatinized template. The studies outlined in this proposal build on these observations to reveal fundamental mechanisms of transcription and how EBV subverts epigenetic regulatory mechanisms to complete its lifecycle. Our first objective (AIM-2A of Project 3 from our P01) will be to visualize EBV lytic RNAs by different stemloops into early versus late mRNAs and visualize them throughout the replication cycle using stem-loop binding domains fused to fluorescent proteins. This will allow us to define the spatial organization of early and late transcription and determine whether late genes arise from EBV replication factories. Our second objective will be to define the templates used for late gene transcription, in particular whether it is distinct from the DNA destined for packaging into virions. Our third objective will be to examine the localization of the EBV DNA polymerase and vPIC components in live-cells undergoing EBV replication to determine if DNA synthesis and late gene transcription are spatially coupled. We expect to determine whether transcription of non-chromatinized DNA in replication compartments is accompanied by cessation of transcription from chromatinized host and viral DNA. Collectively studies will define how EBV subverts normal epigenetic regulatory mechanism to express a substantial fraction of its gene repertoire from unchromatized DNA templates are part of its replication strategy. We expect our results to reveal important insights into the EBV lifecycle and the cellular checkpoints that EBV must circumvent to accomplish this feat.